Toll-like receptors (TLRs) and members of the interleukin-1 receptor (IL-lR) superfamily have crucial roles in host innate immune response against microbial infections. For example, deletion of the TLR4 gene in mice renders them hyporesponsive to lipopolysaccharide (LPS, also known as endotoxin), which is a unique cell-wall component of Gram-negative bacteria. In mammals and other vertebrates, the innate immune response is also important for the activation of the proper adaptive immune response. The TLRs and the IL-1RS share a conserved intra-cellular domain, the Toll/Interleukin-1 Receptor (TIR) domain. The integrity of this domain is required for the signal transduction through these receptors. For example, a single-point mutation in the TIR domain of murine TLR4, P712H, leads to the abrogation of LPS responsiveness, similar to the phenotype of the TLR4-/- mice. Additional mutagenesis studies also confirm the importance of this domain in the signaling process. The signaling pathway of the TLRs and IL-1RS involves many molecules, such as the adapter molecule MyD88, the protein kinase IRAK, TRAF6, and other down-stream mediators. MyD88 is a cytoplasmic protein and it also contains a TIR domain. Ultimately, the signal transduction leads to the activation of the transcription factor NF-kappaB, the stress kinases and the AP-1 transcription factors. TIR domains are believed to be protein-protein interaction modules. Upon receptor activation by ligand binding, an intra-cellular signaling complex is formed via the TIR domains that are present in the receptors and the MyD88 adapter molecule. This is a crucial step in the signal transduction by these receptors. Currently, there is no structural information on any of the TIR domains. The proposed research project will fill this gap in our knowledge on these signal transduction modules. The structural information will be used to understand the biology and biochemistry of the TLRs and the IL-1RS. The information will also be used to identify residues that may be important for the signal transduction. These will form the basis for further mutagenesis, biochemical, and functional studies to assess their importance.